Azoxymethane/Dextran Sodium Sulfate (AOM/DSS) Model of Colorectal Cancer.

Recent progress in developing new vaccination strategies against cancer requires the production of complex and reliable animal models reflecting the complexity of the tumors with their microenvironment. Mice can be considered a good source due to low cost and ease of being genetically modified, inoculated with tumor cell lines or treated by chemicals to induce different cancers. Despite significant limitations in modeling human cancer complexity, preclinical trials conducted in mice can efficiently contribute to understand molecular mechanisms of cancer, to closely resemble and follow carcinogenesis steps impossible to study into humans, and to test new anticancer therapies. In this chapter, we generally describe the different mouse models developed for cancer vaccines' preclinical trials. A particular focus is dedicated to a chemically-induced colorectal cancer model in use in our laboratories.

Changes in Gut Microbiome upon Orchiectomy and Testosterone Administration in AOM/DSS-Induced Colon Cancer Mouse Model.

PURPOSE: Sex hormones are known to affect the gut microbiota. Previously, we reported that endogenous and exogenous testosterone are associated with colorectal cancer (CRC) development and submucosal invasion. In the present study, we investigated whether the gut microbiota is affected by orchiectomy (ORX) and testosterone propionate (TP) administration using an azoxymethane/dextran sulfate sodium (AOM/DSS)-induced CRC mouse model. MATERIALS AND METHODS: Gut microbiota was evaluated by means of 16S rRNA gene sequencing of stool DNA extracted from feces that were obtained at 13 weeks after AOM injection (from 22-week-old animals) and stored in a gas-generating pouch. RESULTS: The increase in microbial diversity (Chao1 and Phylogenetic Diversity index) and Firmicutes/Bacteroidetes (F/B) ratio upon AOM/DSS treatment in ORX mice was significantly decreased by TP supplementation. The ratio of commensal bacteria to opportunistic pathogens was lower in the TP-administered females and ORX mice than in the AOM/DSS group. Opportunistic pathogens (Mucispirillum schaedleri or Akkermansia muciniphila) were identified only in the TP group. In addition, microbial diversity and F/B ratio were higher in male controls than in female and ORX controls. Flintibacter butyricus, Ruminococcus bromii, and Romboutsia timonensis showed similar changes in the male control group as those in the female and ORX controls. CONCLUSION: In conclusion, testosterone determines the dysbiosis of gut microbiota, which suggests that it plays a role in the sex-related differences in colorectal carcinogenesis.

Supplementation with High or Low Iron Reduces Colitis Severity in an AOM/DSS Mouse Model.

The relationship between colitis-associated colorectal cancer (CAC) and the dysregulation of iron metabolism has been implicated. However, studies on the influence of dietary iron deficiency on the incidence of CAC are limited. This study investigated the effects of dietary iron deficiency and dietary non-heme iron on CAC development in an azoxymethane/dextran sodium sulfate (AOM/DSS) mouse model. The four-week-old mice were divided into the following groups: iron control (IC; 35 ppm iron/kg) + normal (NOR), IC + AOM/DSS, iron deficient (ID; <5 ppm iron/kg diet) + AOM/DSS, and iron overload (IOL; approximately 2000 ppm iron/kg) + AOM/DSS. The mice were fed the respective diets for 13 weeks, and the AOM/DSS model was established at week five. FTH1 expression increased in the mice's colons in the IC + AOM/DSS group compared with that observed in the ID and IOL + AOM/DSS groups. The reduced number of colonic tumors in the ID + AOM/DSS and IOL + AOM/DSS groups was accompanied by the downregulated expression of cell proliferation regulators (PCNA, cyclin D1, and c-Myc). Iron overload inhibited the increase in the expression of NF-κB and its downstream inflammatory cytokines (IL-6, TNFα, iNOS, COX2, and IL-1ß), likely due to the elevated expression of antioxidant genes (SOD1, TXN, GPX1, GPX4, CAT, HMOX1, and NQO1). ID + AOM/DSS may hinder tumor development in the AOM/DSS model by inhibiting the PI3K/AKT pathway by increasing the expression of Ndrg1. Our study suggests that ID and IOL diets suppress AOM/DSS-induced tumors and that long-term iron deficiency or overload may negate CAC progression.

Testosterone strongly enhances azoxymethane/dextran sulfate sodium-induced colorectal cancer development in C57BL/6 mice.

Colorectal cancer (CRC) is known to occur more frequently in males than in females, with sex hormones reportedly influencing the development. The purpose of the study was to investigate whether orchiectomy in C57BL/6 male mice reduces colorectal tumorigenesis and whether testosterone administration increases tumorigenesis after orchiectomy in an azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model. Clinical symptoms, including colitis and tumor incidence, were evaluated in the absence or presence of testosterone in AOM/DSS-treated male, as well as orchiectomized (ORX) male and female mice. The levels of serum testosterone and colonic myeloperoxidase, interleukin (IL)-1ß, and IL-6 were measured by ELISA. Target mRNA expression was assessed by quantitative real-time PCR. Orchiectomy significantly diminished the AOM/DSS-induced colitis indices, including disease activity index, colon shortening, and histological severity at week 2, and decreased tumor numbers and incidence rates in the distal part of the colon increased following AOM/DSS administration at week 13; this reduction was reversed by testosterone supplementation. Furthermore, it was confirmed that the ELISA level (MPO and IL-1ß) and the mRNA expression of the inflammatory mediators (COX-2 and iNOS) were maintained at high levels in the tumors of the testosterone-treated group compared with AOM/DSS groups. Interestingly, both endogenous and exogenous testosterone administrations were associated with tumor development (> 2 mm in size) and submucosal invasive cancer. Based on multivariate logistic regression analysis, testosterone was identified as a reasonable hazard factor for the progression of submucosal invasive cancer of the distal colon. In conclusion, endogenous and exogenous testosterone presented a stimulating effect on AOM/DSS-induced colitis and carcinogenicity.

Changes in Microbial Community Composition Related to Sex and Colon Cancer by Nrf2 Knockout.

The frequency of azoxymethane/dextran sulfate sodium (AOM/DSS)-induced carcinogenesis in male mice is higher than that in female mice. Previous studies have reported that 17ß-estradiol inhibits tumorigenesis in males by modulating nuclear factor-erythroid 2-related factor 2 (Nrf2). This study aimed to investigate the changes in mouse gut microbiome composition based on sex, AOM/DSS-induced colorectal cancer (CRC), and Nrf2 genotype. The gut microbiome composition was determined by 16S rRNA gene sequencing fecal samples obtained at week 16 post-AOM administration. In terms of sex differences, our results showed that the wild-type (WT) male control mice had higher alpha diversity (i.e. Chao1, Shannon, and Simpson) than the WT female control mice. The linear discriminant analysis effect size (LEfSe) results revealed that the abundances of Akkermansia muciniphila and Lactobacillus murinus were higher in WT male control mice than in WT female controls. In terms of colon tumorigenesis, the alpha diversity of the male CRC group was lower than that of the male controls in both WT and Nrf2 KO, but did not show such changes in females. Furthermore, the abundance of A. muciniphila was higher in male CRC groups than in male controls in both WT and Nrf2 KO. The abundance of Bacteroides vulgatus was higher in WT CRC groups than in WT controls in both males and females. However, the abundance of L. murinus was lower in WT female CRC and Nrf2 KO male CRC groups than in its controls. The abundance of A. muciniphila was not altered by Nrf2 KO. In contrast, the abundances of L. murinus and B. vulgatus were changed differently by Nrf2 KO depending on sex and CRC. Interestingly, L. murinus showed negative correlation with tumor numbers in the whole colon. In addition, B. vulgatus showed positive correlation with inflammatory markers (i.e. myeloperoxidase and IL-1ß levels), tumor numbers, and high-grade adenoma, especially, developed mucosal and submucosal invasive adenocarcinoma at the distal part of the colon. In conclusion, Nrf2 differentially alters the gut microbiota composition depending on sex and CRC induction.

Nuclear Factor Erythroid 2-related Factor 2 Knockout Suppresses the Development of Aggressive Colorectal Cancer Formation Induced by Azoxymethane/Dextran Sulfate Sodium-Treatment in Female Mice.

Colon tumors develop more frequently in male than in female. Nuclear factor erythroid 2-related factor 2 (Nrf2) plays differential roles in the stage of tumorigenesis. The purpose of this study was to investigate the role of Nrf2 on colitis-associated tumorigenesis using Nrf2 knockout (KO) female mice. Azoxymethane (AOM) and dextran sulfate sodium (DSS)-treated wild-type (WT) and Nrf2 KO female mice were sacrificed at week 2 and 16 after AOM injection. Severity of colitis, tumor incidence, and levels of inflammatory mediators were evaluated in AOM/DSS-treated WT and Nrf2 KO mice. Furthermore, qRT-PCR, Western blot abnalysis, and ELISA were performed in colon tissues. At week 2, AOM/DSS-induced colon tissue damages were significantly greater in Nrf2 KO than in WT mice. At week 16, tumor numbers (> 2 mm size) were significantly lower in both the proximal and distal colon in Nrf2 KO compared to WT. The overall incidences of adenoma/cancer of the proximal colon and submucosal invasive cancer of the distal colon were reduced by Nrf2 KO. The mRNA and protein expression levels of NF-κB-related mediators (i.e., iNOS and COX-2) and Nrf2-related antioxidants (i.e., heme oxygenase-1 and glutamate-cysteine ligase catalytic subunit) were significantly lower in the Nrf2 KO than in WT mice. Interestingly, the protein level of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) was higher in AOM/DSS-treated Nrf2 KO than in WT mice. Our results support the oncogenic effect of Nrf2 in the later stage of carcinogenesis and upregulation of tumor suppressor 15-PGDH might contribute to the repression of colitis-associated tumorigenesis in Nrf2 KO female mice.

17ß-Estradiol strongly inhibits azoxymethane/dextran sulfate sodium-induced colorectal cancer development in Nrf2 knockout male mice.

Nuclear factor erythroid 2-related factor 2 (Nrf2) has dual effects on inflammation and cancer progression depending on the microenvironment. Estrogens have a protective effect on colorectal cancer (CRC) development. The aim of this study was to investigate CRC development in Nrf2 knockout (KO) mice. Azoxymethane (AOM) and dextran sulfate sodium (DSS)-treated wild-type (WT) and Nrf2 KO male mice were sacrificed at weeks 2 and 16 after AOM injection with/without 17ß-estradiol (E2) treatment during week 1. Disease activity index and colon tissue damage at week 2 showed strong attenuation following E2 administration in WT mice but to a lesser extent in Nrf2 KO male mice. At week 16, E2 significantly diminished AOM/DSS-induced adenoma/cancer incidence at distal colon in the Nrf2 KO group, but not in the WT. Furthermore, mRNA or protein levels of NF-κB-related mediators (i.e., iNOS, TNF-α, and IL-1ß) and Nrf2-related antioxidants (i.e., NQO1 and HO-1) were significantly lower in the Nrf2 KO group regardless of E2 treatment compared to the WT. The expression of estrogen receptor beta (ERß) was higher in the Nrf2 KO group than in the WT. In conclusion, estrogen further inhibits CRC by upregulating ERß-related alternate pathways in the absence of Nrf2.

Effects of 17ß-estradiol on colorectal cancer development after azoxymethane/dextran sulfate sodium treatment of ovariectomized mice.

Estrogen is known to have a protective effect in colorectal cancer (CRC) development. Previously, we reported the anti-inflammatory and antitumorigenic effects of 17ß-estradiol (E2) in azoxymethane (AOM)/dextran sulfate sodium (DSS)-treated male mice. The aim of this study was to investigate whether ovariectomy in a female AOM/DSS mouse model increases colorectal tumorigenesis and whether tumorigenesis is reduced by estrogen supplementation after ovariectomy. Clinical symptoms and histological severity of colitis and the levels of inflammatory mediators were evaluated in the colon of AOM/DSS-treated ovariectomized (OVX) mice. The levels of E2, myeloperoxidase (MPO), and NF-κB-dependent cytokines (interleukin (IL)-1ß and IL-6) were measured by ELISA. Furthermore, quantitative real-time (qRT) PCR and Western blot analysis were performed. Ovariectomy did not aggravate AOM/DSS-induced colitis at 2 weeks. At weeks 10 and 16, ovariectomy significantly increased tumor number and incidence rate in only the proximal colon after AOM/DSS treatment (F_AOM/DSS vs OVX_AOM/DSS), and these increases were significantly reduced by E2 supplementation (OVX_AOM/DSS vs OVX_AOM/DSS/E2). However, ovariectomy did not affect CRC development in the distal colon (F_AOM/DSS vs OVX_AOM/DSS). At week 2, E2 administration to AOM/DSS-treated OVX mice attenuated the histological severity of colitis by decreasing the protein and/or mRNA levels of estrogen receptor alpha (ERα) and NF-κB-related mediators (i.e., COX-2, TNF-α, and IL-6) and by enhancing estrogen receptor beta (ERß) and nuclear Nrf2 protein expression and the mRNA expression of related antioxidant enzyme genes (i.e., HO-1, GCLC, GCLM, and NQO1). Endogenous estrogen in females protects against the development of proximal colon cancer, and exogenous E2 replacement in OVX female mice showed protective effects against AOM/DSS-induced colitis and carcinogenesis. The mechanism could involve modulating ERs-, NF-κB- and Nrf2-mediated pathways.

Effect of Estradiol in an Azoxymethane/Dextran Sulfate Sodium-Treated Mouse Model of Colorectal Cancer: Implication for Sex Difference in Colorectal Cancer Development.

PURPOSE: This study demonstrates that estradiol downregulates inflammation and inhibits colorectal cancer (CRC) development in azoxymethane/dextran sulfate sodium (AOM/DSS) mouse model. MATERIALS AND METHODS: AOM/DSS-treated male and female mice were sacrificed at weeks 2, 10, and 16, to assess estrogen effects on colitis and carcinogenesis. Macroscopic and histologic severity of colitis and Western blot and quantitative real-time polymerase chain reaction were evaluated, to measure inflammatory mediators and cytokines. RESULTS: Compared with AOM/DSS-treated male mice (M-AOM/DSS group), AOM/DSS-treated male mice with estradiol administration (M-AOM/DSS+estr group) displayed at week 2 significantly decreased severity of colitis. At weeks 10 and 16, AOM/DSS-treated female mice (F-AOM/DSS group) and the M-AOM/DSS+estr group showed significantly lower tumor multiplicity compared with the M-AOM/DSS group. At week 2, F-AOM/DSS group had a lower level of nuclear factor-κB (NF-κB) expression and higher level of nuclear factor erythroid 2-related factor 2 (Nrf2) expression, compared to the M-AOM/DSS group. At week 2, expression levels of NF-κB and its related mediators decreased in the M-AOM/DSS+estr group, while levels of Nrf2 and Nrf2-related anti-oxidant enzymes increased. In addition, estradiol significantly increased Nod-like receptor protein 3 (NLRP3) inflammasome expressions in AOM/DSS-treated male mice. In contrast, at weeks 10 and 16, Nrf2 and its-related anti-oxidant enzymes and NLRP3 inflammasome were highly expressed in M-AOM/DSS group and in F-AOM/DSS group, who developed cancer. CONCLUSION: The data suggest that estradiol inhibits the initiation of CRC by regulating Nrf2-related pathways. Moreover, these imply the dual role of Nrf2 and NLRP3 inflammasome, including promotion of tumor progression upon tumor initiation.

Effect of Estradiol in an Azoxymethane/Dextran Sulfate Sodium-Treated Mouse Model of Colorectal Cancer: Implication for Sex Difference in Colorectal Cancer Development / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: This study demonstrates that estradiol downregulates inflammation and inhibits colorectal cancer (CRC) development in azoxymethane/dextran sulfate sodium (AOM/DSS) mouse model. MATERIALS AND METHODS: AOM/DSS-treated male and female mice were sacrificed at weeks 2, 10, and 16, to assess estrogen effects on colitis and carcinogenesis. Macroscopic and histologic severity of colitis and Western blot and quantitative real-time polymerase chain reaction were evaluated, to measure inflammatory mediators and cytokines. RESULTS: Compared with AOM/DSS-treated male mice (M-AOM/DSS group), AOM/DSS-treated male mice with estradiol administration (M-AOM/DSS+estr group) displayed at week 2 significantly decreased severity of colitis. At weeks 10 and 16, AOM/DSS-treated female mice (F-AOM/DSS group) and the M-AOM/DSS+estr group showed significantly lower tumor multiplicity compared with the M-AOM/DSS group. At week 2, F-AOM/DSS group had a lower level of nuclear factor-κB (NF-κB) expression and higher level of nuclear factor erythroid 2-related factor 2 (Nrf2) expression, compared to the M-AOM/DSS group. At week 2, expression levels of NF-κB and its related mediators decreased in the M-AOM/DSS+estr group, while levels of Nrf2 and Nrf2-related anti-oxidant enzymes increased. In addition, estradiol significantly increased Nod-like receptor protein 3 (NLRP3) inflammasome expressions in AOM/DSS-treated male mice. In contrast, at weeks 10 and 16, Nrf2 and its-related anti-oxidant enzymes and NLRP3 inflammasome were highly expressed in M-AOM/DSS group and in F-AOM/DSS group, who developed cancer. CONCLUSION: The data suggest that estradiol inhibits the initiation of CRC by regulating Nrf2-related pathways. Moreover, these imply the dual role of Nrf2 and NLRP3 inflammasome, including promotion of tumor progression upon tumor initiation.

Combination Strategy of Quantitative Proteomics Uncovers the Related Proteins of Colorectal Cancer in the Interstitial Fluid of Colonic Tissue from the AOM-DSS Mouse Model.

Quantitative proteome analysis using iTRAQ is an important technique to find disease-related proteins. As an important component of tumor microenvironment, tissue interstitial fluid (TIF) has drawn a great attention for its potential as a source for exploration of the solid tumor biomarkers. On the basis of a mouse model of colorectal cancer (CRC) that was induced by the carcinogenetic reagents, we adopted a quantitative proteome analysis with iTRAQ to discover the CRC-related proteins in the TIFs and with MRM to evaluate the corresponding abundance changes in the individual mouse TIF and serum samples.

Wogonoside prevents colitis-associated colorectal carcinogenesis and colon cancer progression in inflammation-related microenvironment via inhibiting NF-κB activation through PI3K/Akt pathway.

The inflammatory microenvironment has been reported to be correlated with tumor initiation and malignant development. In the previous studies we have found that wogonoside exerts anti-neoplastic and anti-inflammatory activities. In this study, we aimed to further investigate the chemopreventive effects of wogonoside on colitis-associated cancer and delineated the potential mechanisms. In the azoxymethane initiated and dextran sulfate sodium (AOM/DSS) promoted colorectal carcinogenesis mouse model, wogonoside significantly reduced the disease severity, lowered tumor incidence and inhibited the development of colorectal adenomas. Moreover, wogonoside inhibited inflammatory cells infiltration and cancer cell proliferation at tumor site. Furthermore, wogonoside dramatically decreased the secretion and expression of IL-1ß, IL-6 and TNF-α as well as the nuclear expression of NF-κB in adenomas and surrounding tissues. In vitro results showed that wogonoside suppressed the proliferation of human colon cancer cells in the inflammatory microenvironment. Mechanistically, we found that wogonoside inhibited NF-κB activation via PI3K/Akt pathway. In conclusion, our results demonstrated that wogonoside attenuated colitis-associated tumorigenesis in mice and inhibited the progression of human colon cancer in inflammation-related microenvironment via suppressing NF-κB activation by PI3K/Akt pathway, indicating that wogonoside could be a promising therapeutic agent for colorectal cancer.

Discovery of potential colorectal cancer serum biomarkers through quantitative proteomics on the colonic tissue interstitial fluids from the AOM-DSS mouse model.

Quantitative proteomic analysis was performed using iTRAQ to discover colorectal cancer (CRC)-related proteins in tissue interstitial fluids (TIFs). A typical inflammation-related CRC mouse model was generated using azoxymethane-dextran sodium sulfate (AOM-DSS), and TIFs were collected from these mice in four stages during CRC development. Using stringent criteria, a total of 144 proteins displayed changes in their abundances during tumor growth, including 45 that consecutively increased, 17 that consecutively decreased and 82 that changed irregularly. Of these 144 proteins, 24 of the consecutively changed proteins were measured using MRM in individual TIF samples, and 18 were verified. Twelve proteins verified to be consecutively increased in TIFs were examined using MRM to evaluate changes in their abundance in individual mouse serum samples. The abundances of leucine-rich alpha-2-glycoprotein 1 (LRG1), tubulin beta-5 chain (TUBB5) and immunoglobulin J chain (IGJ) were significantly higher in CRC mice than in control mice. Using clinical samples and MRM, we further verified that LRG1 and TUBB5 are potential CRC serum biomarkers. These data demonstrate that coupling dynamic TIF proteomics with targeted serum proteomics in an animal model is a promising avenue for pursuing the discovery of tumor serum biomarkers. BIOLOGICAL SIGNIFICANCE: Colorectal cancer (CRC) is one of the most dangerous diseases worldwide. However, few of CRC biomarkers possess satisfied specificity and sensitivity in clinical practices. Exploration of more CRC biomarkers, especially in serum, is an urgent and also a time-consuming campaign in the CRC study. Our study demonstrates that quantitatively evaluating the phase-dependent proteins in colonic tissue interstitial fluids from AOM-DSS mice is a feasible and effective way for exploration of the CRC-related proteins and the potential serum biomarkers. We identified two proteins, LRG1 and TUBB5, which may be practicable in human clinical samples as CRC serum biomarkers. To sum up, this study provides a novel angle to explore the critical factors in tumorigenesis and a new pipeline for potential serum biomarker discovery and verification.